Solid-State Formation of a Potential Melphalan Delivery Nanosystem Based on β-Cyclodextrin and Silver Nanoparticles

Melphalan (Mel) is an antineoplastic widely used in cancer and other diseases. Its low solubility, rapid hydrolysis, and non-specificity limit its therapeutic performance. To overcome these disadvantages, Mel was included in β-cyclodextrin (βCD), which is a macromolecule that increases its aqueous solubility and stability, among other properties. Additionally, the βCD–Mel complex has been used as a substrate to deposit silver nanoparticles (AgNPs) through magnetron sputtering, forming the βCD–Mel–AgNPs crystalline system. Different techniques showed that the complex (stoichiometric ratio 1:1) has a loading capacity of 27%, an association constant of 625 M−1, and a degree of solubilization of 0.034. Added to this, Mel is partially included, exposing the NH2 and COOH groups that stabilize AgNPs in the solid state, with an average size of 15 ± 3 nm. Its dissolution results in a colloidal solution of AgNPs covered by multiple layers of the βCD–Mel complex, with a hydrodynamic diameter of 116 nm, a PDI of 0.4, and a surface charge of 19 mV. The in vitro permeability assays show that the effective permeability of Mel increased using βCD and AgNPs. This novel nanosystem based on βCD and AgNPs is a promising candidate as a Mel nanocarrier for cancer therapy.


Introduction
Melphalan (Mel) is a bifunctional alkylating agent widely used at the clinical level in the treatment of multiple types of cancer and other diseases [1][2][3][4]. The effectiveness of killing cancer cells of this drug is based on the ability to react extensively with DNA, RNA, and proteins to form interstrand DNA crosslinks, inducing multiple kinds of molecular lesions [5][6][7]. Nevertheless, Mel presents serious therapeutic disadvantages related to its non-specificity, the resistance of tumor cells, and the need for increasingly higher doses during treatment, generating adverse effects that include leukopenia, mucositis, and diarrhea [8,9]. In this sense, the administration of Mel could be optimized using nanotechnology [10][11][12][13][14][15][16][17]. Specifically, silver nanoparticles (AgNPs) have been used for the loading and transport of antitumor drugs, showing a synergistic effect with these drugs and a site-specific action, which would reduce the administered dose and even reduce the toxicity in healthy cells [18][19][20]. This research field is controversial; therefore, it is necessary to expand on this point. nanosystem size, among others, were included. In addition, the effective permeability of Mel in βCD and with AgNPs was evaluated in an in vitro membrane model. Our research is the first report of Mel complexation in βCD and its interaction with AgNPs in a solid state and surfactant-free, where both components work to solve some therapeutic drawbacks of this drug and as a potential nanocarrier. were dissolved, forming the new βCD-Mel-AgNPs colloidal nanosystem, which was characterized using dynamic light scattering, zeta potential, and transmission electron microscopy. The study of relevant pharmaceutical parameters, such as association constant, loading capacity, and nanosystem size, among others, were included. In addition, the effective permeability of Mel in βCD and with AgNPs was evaluated in an in vitro membrane model. Our research is the first report of Mel complexation in βCD and its interaction with AgNPs in a solid state and surfactant-free, where both components work to solve some therapeutic drawbacks of this drug and as a potential nanocarrier.

Formation of the Complex in Solid State
The diffraction patterns of the βCD, Mel, and the complex formed were obtained using PXRD (see Figure 2). The formation of the complex in the solid state causes the disappearance of the Mel peaks, especially in the region above 25° 2θ, maintaining a peak corresponding to βCD at 2θ angle of approximately 12°. The crystal packing arrangement of the complex was different from that of its pure species and represents a new crystalline phase, confirming the effective inclusion of the drug in the βCD matrix. Three intense peaks were observed at approximately 4°, 12° and 20° 2θ in the βCD-Mel pattern. It has been reported that the formation of complexes with a P21-type structure for βCD matrices hosting aromatic rings at 1:1 (host-guest) molar ratios also exhibit three intense peaks at equal locations. In addition, the geometrical arrangement and thus the respective diffraction pattern varies according to the type of molecular structure of the aromatic guest, according to the region partially exposed to the outside of the βCD cavity and to the water molecules contributing to the new crystalline packing [48][49][50]. Regarding the physical mixture of pure components, (Figure 2d), it was observed that its diffractogram corresponds to a superposition between the traces of the pure species.

Formation of the Complex in Solid State
The diffraction patterns of the βCD, Mel, and the complex formed were obtained using PXRD (see Figure 2). The formation of the complex in the solid state causes the disappearance of the Mel peaks, especially in the region above 25 • 2θ, maintaining a peak corresponding to βCD at 2θ angle of approximately 12 • . The crystal packing arrangement of the complex was different from that of its pure species and represents a new crystalline phase, confirming the effective inclusion of the drug in the βCD matrix. Three intense peaks were observed at approximately 4 • , 12 • and 20 • 2θ in the βCD-Mel pattern. It has been reported that the formation of complexes with a P2 1 -type structure for βCD matrices hosting aromatic rings at 1:1 (host-guest) molar ratios also exhibit three intense peaks at equal locations. In addition, the geometrical arrangement and thus the respective diffraction pattern varies according to the type of molecular structure of the aromatic guest, according to the region partially exposed to the outside of the βCD cavity and to the water molecules contributing to the new crystalline packing [48][49][50]. Regarding the physical mixture of pure components, (Figure 2d), it was observed that its diffractogram corresponds to a superposition between the traces of the pure species.

Stability of the Complex in Solution
1 H-NMR was conducted to study the conformation and stoichiometry of βCD-Mel and to evaluate the stability of Mel included in the complex. The latter was chosen because, in therapy, Mel has poor solubility in water and is unstable on reconstitution and dilution [44]. In 1978, Chang et al. proposed that the hydrolysis of Mel occurs in three phases where chloroethyls, one by one, are transformed into hydroxyethyls by a threecomponent cyclic intermediate mechanism with the tertiary amine. This process is completed after 8 h [37].
Once the complex was formed in the solid state, it was dissolved, 1 H-NMR spectrum of βCD-Mel in solution was registered and then compared with the free βCD and Mel spectra (see Figure 3).

Stability of the Complex in Solution
1 H-NMR was conducted to study the conformation and stoichiometry of βCD-Mel and to evaluate the stability of Mel included in the complex. The latter was chosen because, in therapy, Mel has poor solubility in water and is unstable on reconstitution and dilution [44]. In 1978, Chang et al. proposed that the hydrolysis of Mel occurs in three phases where chloroethyls, one by one, are transformed into hydroxyethyls by a three-component cyclic intermediate mechanism with the tertiary amine. This process is completed after 8 h [37].
Once the complex was formed in the solid state, it was dissolved, 1 H-NMR spectrum of βCD-Mel in solution was registered and then compared with the free βCD and Mel spectra (see Figure 3).

Stability of the Complex in Solution
1 H-NMR was conducted to study the conformation and stoichiometry of βCD-Mel and to evaluate the stability of Mel included in the complex. The latter was chosen because, in therapy, Mel has poor solubility in water and is unstable on reconstitution and dilution [44]. In 1978, Chang et al. proposed that the hydrolysis of Mel occurs in three phases where chloroethyls, one by one, are transformed into hydroxyethyls by a threecomponent cyclic intermediate mechanism with the tertiary amine. This process is completed after 8 h [37].
Once the complex was formed in the solid state, it was dissolved, 1 H-NMR spectrum of βCD-Mel in solution was registered and then compared with the free βCD and Mel spectra (see Figure 3).   Table 1 shows the chemical shifts of βCD-Mel compared to its pure species. The shifts of the 2 /6 , 3 /5 protons of the ring and the 1 and 2 protons of the drug chain towards lower fields demonstrate interaction with the CD matrix; while the chemical shifts of the H -3 a and b protons of Mel suggest interactions with the OH groups present on the surface of the βCD cavity. The internal H-3, H-5, and H-6 matrix protons demonstrate that the drug remains included. The OH-6 groups move to higher fields due to the proximity of electronegative groups such as COOH and NH 2 that are close to one of the CD openings. Considering the changes observed in Mel upon hydrolyzing and the signals observed in the spectrum, it is confirmed that through the formation of the complex, the drug can remain stable within the matrix, considerably increasing its applicability in therapy. Integrating the proton signals of βCD and Mel in the 1 H-NMR spectrum of the complex allowed for the determination of the host:guest stoichiometric ratio in solution, according to a methodology used for different CD complexes [51,52]. The integrations of the H-1 proton of the βCD with H -2 /6 and H -3/5 protons of the aromatic ring of Mel were compared. The results show that the complex was formed in a 1:1 stoichiometric ratio, being relevant for potential applications (more details are given in the Supplementary Materials, Section S1).

Drug Loading and Complexation Efficiency
In pharmaceutical concepts, knowing the stoichiometric ratio allows us to obtain important parameters for potential formulation production [13][14][15][16]53]. The 1:1 drug/βCD molar ratio means that the 0.75 g of cyclodextrin can load 0.20 g of drug, so the loading capacity of the matrix was 27%. The solubility of βCD in water is 18.5 g/L [54], so to completely dissolve the inclusion complex, about 40.5 mL is needed, where 200 mg of the drug would be found. This means that the formation of the complex increases the practical solubility of Mel almost five times.
On the other hand, the phase solubility method allows knowing parameters such as the degree of solubilization, association constant, and complexation efficiency [53] (further details of the procedure and figures are provided in the Supplementary Materials, Section S2). Linear increases in the aqueous solubilities of Mel versus βCD used were observed, with a 0.034 degree of solubilization. In general, the recommendable K a values of drug:CD complexes are between 50 and 2000 M −1 , because at values below 50 M −1 , the pharmaceutical formulation may be limited by its low stability, not favoring a controlled release, e.g., an early liberation into the blood and not into the desired tissue. On the other hand, at high K a values (>2000 M −1 ), the ability to release the drug by gradient or by stimulus can be affected by the high degree of inclusion of the drug in the βCD cavity, excessively delaying its pharmacokinetics, among other possible therapeutic drawbacks [55,56]. The K a of βCD-Mel was 625 M −1 , being located in an adequate range, with stable host-guest interactions, which prevent its early release and could allow its delivery to the site of action. Furthermore, the complexation efficiency can be used to probe the feasibility of using cyclodextrins in the formulation of drugs. In the βCD-Mel system, the complexation efficiency value was 0.035, being suitable for a potential formulation based on this complex.

Molecular Interaction and Geometry of the Inclusion Complex
Whereas the formation, stability, and loading parameters of Mel on the complex were in the desired range, for the formation of the nanovehicle by stabilizing AgNPs directly on its surface using βCD, it was necessary to know the availability of adequate exposed functional groups. The rotational overhauser enhancement spectroscopy (ROESY) technique enables observation of interactions between hydrogen nuclei of non-chemically bonded structures separated by a maximum distance of 5 Å; therefore, it is possible to determine in solution the geometrical structure of supramolecular complexes accurately [51,52]. The ROESY spectra of the βCD-Mel complex are shown in Figure 4a,b. A model showing the proposed geometry of βCD-Mel is shown in Figure 4c (full ROESY spectra are shown in the Supplementary Materials, Section S3).
Whereas the formation, stability, and loading parameters of Mel on the complex w in the desired range, for the formation of the nanovehicle by stabilizing AgNPs direc on its surface using βCD, it was necessary to know the availability of adequate expos functional groups. The rotational overhauser enhancement spectroscopy (ROESY) tec nique enables observation of interactions between hydrogen nuclei of non-chemica bonded structures separated by a maximum distance of 5 Å ; therefore, it is possible determine in solution the geometrical structure of supramolecular complexes accurat [51,52]. The ROESY spectra of the βCD-Mel complex are shown in Figure 4a  This indicates that the aromatic ring is located in the wider region of the matrix. The ex orientation of the drug can be elucidated from the cross-peaks observed between the ternal protons of βCD with the H'-3a proton of Mel ( Figure 4b). This reveals that Me exposed toward the major opening, and thus, the chloroethyl chains are oriented towa the minor opening of the matrix. These results explain the chemical shifts of Mel in t 1 H-NMR spectrum, where shifts towards lower fields of the OH-6 and the H'-1″ and H 2″ protons were observed. This confirms that it is the interaction of the primary hydrox groups of βCD with the chloroethyl chains of Mel that causes this effect. Accordingly, the dimensions of both molecules, the NMR and ROESY result strongly suggests that t COOH and NH2 functional groups of Mel are oriented outside the cavity of the matr thus making it a suitable candidate to stabilize AgNPs.

Deposition of Silver Nanoparticles on the βcyclodextrin-Melphalan Complex
AgNPs were obtained and deposited using the physical method of magnetron sp tering in vacuum. The technique consists of the detachment of atoms from a thin meta film, which agglomerate and stabilize on a substrate, reaching a nanometric size [1 15,57,58]. As we have confirmed, Mel was partially included within βCD, exposing t amine and carboxylic acid functional groups towards one of its openings. This arrang ment allowed the solid-state complexes to act as substrates and the crystalline faces th expose these functional groups to interact specifically with silver atoms allowing its acc mulation, forming, and stabilizing nanoparticles. Figure 5a shows a schematic represe tation of this process. The new βCD-Mel-AgNPs crystalline system was characterized u ing UV-vis diffuse reflectance spectroscopy, SEM, EDX, and FE-SEM. Figure 5b cor sponds to the absorbance spectra of AgNPs deposited on the βCD-Mel complexes. T  (Figure 4a). This indicates that the aromatic ring is located in the wider region of the matrix. The exact orientation of the drug can be elucidated from the cross-peaks observed between the internal protons of βCD with the H -3a proton of Mel (Figure 4b). This reveals that Mel is exposed toward the major opening, and thus, the chloroethyl chains are oriented toward the minor opening of the matrix. These results explain the chemical shifts of Mel in the 1 H-NMR spectrum, where shifts towards lower fields of the OH-6 and the H -1 and H -2 protons were observed. This confirms that it is the interaction of the primary hydroxyl groups of βCD with the chloroethyl chains of Mel that causes this effect. Accordingly, by the dimensions of both molecules, the NMR and ROESY result strongly suggests that the COOH and NH 2 functional groups of Mel are oriented outside the cavity of the matrix, thus making it a suitable candidate to stabilize AgNPs.

Deposition of Silver Nanoparticles on the βcyclodextrin-Melphalan Complex
AgNPs were obtained and deposited using the physical method of magnetron sputtering in vacuum. The technique consists of the detachment of atoms from a thin metallic film, which agglomerate and stabilize on a substrate, reaching a nanometric size [13][14][15]57,58]. As we have confirmed, Mel was partially included within βCD, exposing the amine and carboxylic acid functional groups towards one of its openings. This arrangement allowed the solid-state complexes to act as substrates and the crystalline faces that expose these functional groups to interact specifically with silver atoms allowing its accumulation, forming, and stabilizing nanoparticles. Figure 5a shows a schematic representation of this process. The new βCD-Mel-AgNPs crystalline system was characterized using UV-vis diffuse reflectance spectroscopy, SEM, EDX, and FE-SEM. Figure 5b corresponds to the absorbance spectra of AgNPs deposited on the βCD-Mel complexes. The maximum absorbance peak in the SPR of the nanoparticles was located at 450 nm (see the UV-vis spectra of Mel in solid state in the Supplementary Materials, Section S4).  It has been reported that AgNPs with an average diameter of 10 nm show an SP with a maximum absorbance peak at 400 nm [59]. There are reports of AgNPs formatio on different substrates using this method. In these studies, it is argued that the bath chromic shift of the band above 400 nm is probably due to an increase in the size of t formed nanoparticles (above 10 nm), to the change in the dielectric environment or to a interparticle plasmon coupling [57,[60][61][62].   It has been reported that AgNPs with an average diameter of 10 nm show an SPR with a maximum absorbance peak at 400 nm [59]. There are reports of AgNPs formation on different substrates using this method. In these studies, it is argued that the bathochromic shift of the band above 400 nm is probably due to an increase in the size of the formed nanoparticles (above 10 nm), to the change in the dielectric environment or to an interparticle plasmon coupling [57,[60][61][62].  It has been reported that AgNPs with an average diameter of 10 nm show an S with a maximum absorbance peak at 400 nm [59]. There are reports of AgNPs format on different substrates using this method. In these studies, it is argued that the bat chromic shift of the band above 400 nm is probably due to an increase in the size of formed nanoparticles (above 10 nm), to the change in the dielectric environment or to interparticle plasmon coupling [57,[60][61][62]. Figure 6 corresponds to SEM images showing directly the nanoparticles depos on crystals of the complex, together with an elemental analysis performed on a sectio the crystal using EDX (see more FE-SEM images and EDX table information in Sup mentary Materials, Section S5).  The phenomenon of the inclusion of molecules on βCD generates crystalline arrangements different from that of their pure species. In this case, it was observed that the crystalline morphology of the βCD-Mel complex is different with respect to the pure compounds βCD and Mel (see SEM images information in Supplementary Materials, Section S6).
This corroborates what was previously discussed in the analysis of the samples by XRD. Figure 6a,b shows a high concentration of AgNPs of homogeneous size, preferentially accumulated on one side of the crystal, where NH 2 and COOH functional groups of Mel are exposed, allowing its stabilization and confirming that the phenomenon of aggregation is widely suppressed. The EDX spectra obtained from the surface area analyzed show the atomic and weight elemental composition of the sample (see Figure 6c). The main components were carbon and oxygen; chlorine is also included, coming from the drug, and silver from the nanoparticles that remain stable in the solid state on the surface of the crystals.

Formation of βcyclodextrin-Melphalan-Silver Nanoparticles Nanosystem
The βCD-Mel crystals with deposited AgNPs were solubilized in water, promoting the formation of a colloidal solution of AgNPs stabilized by the βCD-Mel complex, as schematized in Figure 7a. The new βCD-Mel-AgNPs colloidal nanosystem was characterized by DLS, zeta potential, and TEM. The results are summarized in Table 2. The phenomenon of the inclusion of molecules on βCD generates crystalline a ments different from that of their pure species. In this case, it was observed that th talline morphology of the βCD-Mel complex is different with respect to the pur pounds βCD and Mel (see SEM images information in Supplementary Materials, S6).
This corroborates what was previously discussed in the analysis of the samp XRD. Figure 6a,b shows a high concentration of AgNPs of homogeneous size, pr tially accumulated on one side of the crystal, where NH2 and COOH functional gro Mel are exposed, allowing its stabilization and confirming that the phenomenon of gation is widely suppressed. The EDX spectra obtained from the surface area an show the atomic and weight elemental composition of the sample (see Figure 6 main components were carbon and oxygen; chlorine is also included, coming fr drug, and silver from the nanoparticles that remain stable in the solid state on the of the crystals.

Formation of βcyclodextrin-Melphalan-Silver Nanoparticles Nanosystem
The βCD-Mel crystals with deposited AgNPs were solubilized in water, pro the formation of a colloidal solution of AgNPs stabilized by the βCD-Mel comp schematized in Figure 7a. The new ꞵCD-Mel-AgNPs colloidal nanosystem was terized by DLS, zeta potential, and TEM. The results are summarized in Table 2.   The solvation sphere diameter of the nanosystem was 116 nm with a polydispersity of 0.4, characteristic of βCD-based nanosystems [12,16,17,[63][64][65]. Therefore, the dissolution process causes the metal nanoparticles to be surrounded by multiple layers of the complex, as has been previously reported for analogous nanosystems using gold nanoparticles [13][14][15]. In addition, the drug remains partially included in the cavity of the βCD, and its functional groups interact with the surface of Ag atoms. The above was confirmed by the positive surface charge of the βCD-Mel-AgNPs colloidal nanosystem, which was attributed to the NH 2 groups of Mel [66][67][68]. The size of spherical AgNPs was determined using micrographs obtained by TEM (see Figure 7b). A high concentration of AgNPs very close to each other was observed, homogeneous in size and shape, mostly in the range of 10 to 20 nm. Through the analysis of the respective histogram, an average diameter of 15 ± 3 nm was reported. The difference between the TEM size and the hydrodynamic diameter is explained by the multiple layers of βCD-Mel, which cover the nanoparticles and maintain the stability of the colloid. Interestingly, for metal nanoparticles above 5 nm, the size is not a critical parameter in the toxicity profile of a nanosystem, since it has no disruptive effects on healthy cells and can be rapidly excreted by the body [24,69]. Once they accumulate in fenestrated tissue, such as tumors, the expected long-term toxicity of AgNPs is due to the oxidative release of Ag + that could act synergistically for potential treatment [26]. Accordingly, the average size of the nanoparticles and the hydrodynamic diameter of the βCD-Mel-AgNPs colloidal nanosystem new open the way to possible applications in therapy, in addition to being a stable nanocarrier for Mel. In this sense, one of the relevant tests to validate this new system as a nanocarrier in solution is to evaluate its permeability in membranes. For this, studies using the PAMPA method were performed.

Permeability Assays on Artificial Membranes
The PAMPA method is used to predict the passive permeability of biologically relevant species, such as drugs, across the membrane. The assay is performed on a donor and acceptor plate, in which the ability of a compound to diffuse between the two plates separated by a phosphatidylcholine filter is measured, which acts as a lipid membrane and simulates the lipid bilayer of different types of cells. Since artificial membranes do not contain active transport systems, only passive diffusion can be studied [70,71]. Cyclodextrins can enhance the permeability of drugs through an artificial membrane under certain conditions. However, cyclodextrin-based complexes increase the total amount of drug in the solution and the concentration gradient. The molecule excess can decrease penetration, especially lipophilic drugs that could permeate the membrane. Other factors such as agitation speed, pH, type of cyclodextrin, or the association constant of each inclusion complex are variables to be considered [72,73]. Figure 8 shows the effective permeability of Mel, βCD-Mel complex, and βCD-Mel-AgNPs colloidal nanosystem analyzed by PAMPA. The controls used behaved as expected, indicating that the membranes were well constructed. In the case of Mel, as part of the complex, its effective permeability increased with respect to Mel alone, which was consistent with the role of βCD, maintaining drug stability and, in turn, altering membrane properties [74,75]. In addition, Mel, in the presence of AgNPs, also increases its effective permeability with respect to Mel alone, probably because the colloid is surrounded by multiple layers of the βCD-Mel complex [76,77]; however, it does not exceed the permeability of the complex. This is explained by the interaction of the NH 2 functional group of Mel with the surface of the AgNPs, decreasing their migration into the membrane. The foregoing suggests that AgNPs maintain the stability and composition of the nanosystem under the experimental conditions studied, which qualifies the system as a promising candidate for site-directed drug transport [18,26,[78][79][80] since it would avoid its non-specificity or early action in healthy cells during systemic circulation. On the other hand, it conditions the nanocarrier to release the drug through an activation with external stimuli to the biological environment, such as laser irradiation [11,15,17,19,47,81]. foregoing suggests that AgNPs maintain the stability and composition of the nanosystem under the experimental conditions studied, which qualifies the system as a promising candidate for site-directed drug transport [18,26,[78][79][80] since it would avoid its non-specificity or early action in healthy cells during systemic circulation. On the other hand, it conditions the nanocarrier to release the drug through an activation with external stimuli to the biological environment, such as laser irradiation [11,15,17,19,47,81].

Synthesis of the βCD-Mel Complex
To synthesize the βCD-Mel complex, the saturated solutions method was employed [13][14][15]17,82,83]. In total, 0.2025 g of Mel were dissolved in ethanol and then added to a solution of βCD (0.7530 g dissolved in water) at 4 °C with gentle and constant stirring, allowing the temperature to rise slowly up to room temperature. The new solution was kept motionless under a hood for one week until the evaporation of the solvents. Finally, small crystals precipitated at the bottom of the crystallizer were extracted and washed with a 50% v/v solution (ethanol/water) at 4 °C and vacuum filtered for one hour in a kitasate with a Büchner funnel equipped with a double layer of filter paper, to remove the excesses of cyclodextrin, drug or solvents that moisten the complex. The crystals were then pulverized and dried in a vacuum line to remove the water or ethanol, which may remain occluded in the powder, then stored in amber vials with a Teflon seal. The new

Synthesis of the βCD-Mel Complex
To synthesize the βCD-Mel complex, the saturated solutions method was employed [13][14][15]17,82,83]. In total, 0.2025 g of Mel were dissolved in ethanol and then added to a solution of βCD (0.7530 g dissolved in water) at 4 • C with gentle and constant stirring, allowing the temperature to rise slowly up to room temperature. The new solution was kept motionless under a hood for one week until the evaporation of the solvents. Finally, small crystals precipitated at the bottom of the crystallizer were extracted and washed with a 50% v/v solution (ethanol/water) at 4 • C and vacuum filtered for one hour in a kitasate with a Büchner funnel equipped with a double layer of filter paper, to remove the excesses of cyclodextrin, drug or solvents that moisten the complex. The crystals were then pulverized and dried in a vacuum line to remove the water or ethanol, which may remain occluded in the powder, then stored in amber vials with a Teflon seal. The new βCD-Mel complex was characterized by powder X-ray diffraction (PXRD), NMR of one ( 1 H) and two (ROESY) dimensions, UVvis spectroscopy for calculating the loading capacity, degree of solubilization, complexation efficiency, and association constant. The complex was also used for a permeability study called PAMPA (parallel artificial membrane permeability assay).

Formation of Silver Nanoparticles
AgNPs were obtained using magnetron sputtering under a high vacuum, depositing them onto crystalline powder of the βCD-Mel complex acting as a substrate [57,[84][85][86]. In total, approximately 20 mg of this complex was dispersed on a glass slide, which was placed inside the chamber of the equipment, together with a silver foil serving as a cathode. The chamber was evacuated to 0.5 mbar; then, Argon was injected together with a current of 25 mA to ionize the gas, which hit the metal foil, releasing silver atoms that afterward were deposited and stabilized on the complex. The accumulation of atoms on specific crystalline faces of βCD-Mel forms AgNPs in a process that lasts for 20 s. βCD-Mel-AgNPs was characterized by UV-vis spectroscopy, field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), zeta potential, transmission electronic microscopy (TEM), and PAMPA.

Powder X-ray Diffraction (PXRD)
The analysis of the complex and pure species βCD and Mel was performed using a Siemens D-5000 diffractometer with graphite-monochromated Cu K-α radiation at 40 kV and 30 mA with a wavelength of 1.540598 Å.

1 H-NMR and ROESY
Measurements for the complex and pure species βCD and Mel were performed at 300 K in DMSO-d 6 (99.99%) on a Bruker Advance 400 MHz superconducting NMR spectrometer. All 2D NMR spectra were acquired using pulsed-field gradient-selected methods during a mixing time of 12 h. Additionally, the stoichiometry of the complex was evaluated using the integration of the signals of the protons of βCD and Mel in the spectrum of βCD-Mel.

Loading Capacity
The loading capacity of the βCD-Mel was calculated from the weights of βCD and drug obtained using Equation (1) [87].

Loading capacity =
Weight o f drug in βCD Weight o f βCD × 100 (1)

Phase Solubility Studies
Studies were performed following the Higuchi and Connors method [53]. The Beer-Lambert law was used to quantify Mel, considering its absorbance at 301 nm. First, known concentrations (C) of Mel were measured by UV-vis. From the UV-vis spectra, the absorbance maxima at 301 nm (A max ) were extracted. The slope from the A max vs. C graph, corresponded to the ε of the drug (For details, graphs, and tables, see Supplementary Materials, Section S2). Then, the βCD concentration versus the loaded Mel concentration (calculated by Beer-Lambert law) was plotted. The value of the slope of the graphs related to the amount of βCD added to the amount of solubilized drug, indicating the degree of solubilization. The degree of solubilization was used to calculate the association constant (K a ) and complexation efficiency of the βCD-Mel system using Equations (2) and (3), respectively [16].
Complexation e f f iciency = K a(1:1) 12 of 17 [C o ] corresponds to the concentration of the free drug in the absence of βCD.

UV-Vis Spectroscopy in Solid State
The AgNPs deposited onto βCD-Mel were characterized using UV-vis spectroscopy in a solid state [88][89][90]. First, the diffuse reflectance was measured using a Shimadzu UV 2450 spectrophotometer with barium sulfate as a baseline. In addition, the spectrum of the complex was used as a second baseline. Then, the absorbance of the AgNPs was determined using the Kubelka-Munk transformation.

Dynamic Light Scattering and Zeta Potential Measurements
Dynamic light scattering and zeta potential measurements were performed at 25 • C using a Zetasizer model Nano ZS. The size distribution of the samples was determined from the results of the intensity distribution values using the cumulant method. The Smoluchowski approximation was used to calculate zeta potentials based on measured electrophoretic mobility.

Transmission Electron Microscopy
TEM images were obtained using a JEOL JEM 1200 EX II instrument. The samples with AgNPs were prepared by dispersing approximately 0.5 mg in 100 µL of isopropanol (30%). Then, 20 µL of the solution was deposited onto a copper grid with a continuous film of Formvar, the excess solution was removed, and the grid was dried. The acceleration voltage used was 80 kV. More than two thousand nanoparticles were counted to produce the size distribution histogram. The average diameter was obtained from the peak of the Gaussian fit performed on the graph.

Parallel Artificial Membrane Permeability Assay (PAMPA)
The PAMPA studies require two types of well, a donor and an acceptor (Transwell plates); in addition, the acceptor well has a semipermeable membrane of PVDF (polyvinylidene fluoride). On the PVDF membranes were deposited 4.0 µL of a solution of phosphatidylcholine in dodecane (20 mg/mL) for 5 min until complete evaporation of the organic solvent. Subsequently, 300 µL of PBS (pH = 7.4) was added. In the donor well were deposited 300 µL of each sample dissolved in PBS, which are: Mel, βCD-Mel, and βCD-Mel-AgNPs (all concentration data in Supplementary Materials, Section S7). As positive and negative controls, a solution of Thiopental (10 mg in 100 mL of PBS 10 mM) and a solution of Evans Blue (200 mg in 100 mL of PBS 10 mM) were used, respectively. After loading the donor well with the samples and the controls, and the acceptor well with PBS, they were assembled, covered, and sealed using parafilm. Each PAMPA was performed in triplicate (n = 3) for 24 h at 37 • C with constant agitation to 280 rpm.
The effective permeability (Pe) was determined by the following formula: P e = −218.3 t · log 1 − 2 · C A (t) C D (0) · 10 −6 cm/s (4) t = measurement time in hours. CA = concentration in acceptor plate at time t. CD = concentration in donor plate at time zero.
To calculate the concentrations in the acceptor plate, the absorbances of the different plates were measured using a UV-vis spectrophotometer and the respective calibration curve of Mel in PBS [70,71].

Conclusions
In summary, a βCD-Mel crystalline solid complex was formed reproducibly in a 1:1 molar ratio. The hydrophobic cavity of βCD allowed the inclusion of Mel, which prevents hydrolysis and subsequent degradation of the drug. The exhaustive characterization of the complex confirmed that Mel partially exposes the -NH 2 and COOH functional groups, allowing the crystals to act as a substrate for the stabilization of silver atoms, and the consequent formation of AgNPs using magnetron sputtering. In turn, the solubilization of the βCD-Mel-AgNPs crystalline system generates a colloidal solution, where the AgNPs are covered in multiple layers by the complex. The loading capacity, the stability of the drug, and the association constant of the complex, together with the size achieved of the nanoparticles and the effective permeability examined for βCD-Mel and βCD-Mel-AgNPs are promising results that validate this nanosystem as a new drug nanocarrier for Mel. Accordingly, the effective and stable Mel loading in this new nanosystem based on βCD and AgNPs shows potential as a novel alternative to traditional cancer therapeutics.
As a future perspective, evaluating the synergistic effect of the components of the nanosystem, together with studies of cytotoxicity and laser-triggered controlled release of Mel in in vitro and in vivo models, are considered necessary to position this nanosystem as a useful tool in drug delivery applied to cancer.

Conflicts of Interest:
The authors declare no conflict of interest.